JPS6334160B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to novel anthracycline antibiotics that are antitumor substances and methods for producing them. Conventionally, among anthracycline antibiotics, for example, adriamycin (see US Pat. No. 3,590,028) and aclacinomycin A (Japanese Patent Publication No. 51
It is known that it not only shows strong activity against experimental tumors, but also shows strong antitumor properties in clinical trials, and is used as a useful anticancer agent. The present inventors conducted research to develop more useful anthracycline antibiotics, and In the formula, when R ¹ represents a hydroxyl group, R ² is the formula In the sugar residue of , R ³ represents a hydrogen atom; when R ¹ and R ² represent a hydrogen atom, R ³ represents the above sugar residue. They discovered that this compound also exhibits strong activity against tumors in experimental animals, and completed the present invention. The present inventors found that among the compounds represented by formula (), the following formula The compound shown is β-isoromycinone.
Name it RDC and use the following formula The compound represented by is named α ₂ -rhodomycinone RDC and proposed by Tsutsumi. The above β-isorodomycinone RDC and α ₂ -rhodomycinone RDC of the present invention have strong therapeutic effects against murine leukemia (L1210) as described below, but also have excellent therapeutic effects against various other animal experimental tumors. It is expected to have an anticancer effect and has minimal toxicity, so it can be advantageously used as an anticancer agent. The preparation of the compounds of the present invention is based on the formula In the formula, R ¹ represents a hydrogen atom or a hydroxyl group,
Anthracyclinone (hereinafter, a compound in which R ¹ represents a hydrogen atom is referred to as α ₂ -rhodomycinone and a compound in which R ¹ represents a hydroxyl group is referred to as β-isorodomycinone) is an anthracyclinone represented by the formula ().
A mutant strain having the ability to convert R ¹ into α ₂ -rhodomycinone RDC represented by the corresponding formula (-b) and β-isoromycinone RDC represented by the formula (-a) was cultured in a nutrient medium. This can be carried out by adding α ₂ -rhodomycinone or β-isothrodomycinone to the medium or the culture, and collecting the produced α ₂ -rhodomycinone RDC or β-isoromycinone RDC. According to the present invention, the mutant strain having the above-mentioned conversion ability is the above-mentioned aclacinomycin A
Streptomyces galilaeus MA144-M1, which is known as a producing bacterium (see Publication No. 34915)
(ATCC31133 or FERMP-2455), a mutant strain Streptomyces galilaeus MA144 that is non-antibiotic producing and has the above-mentioned conversion ability.
-M1KE303 strain (FERMP-4808) is preferably used, but any strain belonging to any genus can be used as long as it has the above-mentioned conversion ability.
Regarding the isolation method and mycological description of the KE303 strain, see Japanese Patent Application Laid-open No. 15299/1983). Cultivation of the strain or production of the compound of the present invention includes:
First, culture on agar slant medium (yeast extract 0.3%, soluble starch 1.0%, agar 1.5%, pH 7.2) at 6-7℃.
Strains with the above-mentioned transforming ability stored in
KE303 strain) was inoculated into a nutrient medium commonly used for culturing microorganisms belonging to the genus Streptomyces, such as a normal liquid medium consisting of starch, glucose, an organic nitrogen source, and inorganic salts, and incubated at 25-32°C for 1-3 hours. Prepare the seed mother by culturing with shaking for several days. Next, 1 to 3% of the above seed mother was inoculated into a normal liquid medium, such as a medium consisting of sucrose, glucose, soybean flour, and inorganic salts, and cultured with shaking at 25 to 32°C for 15 to 48 hours. To the culture solution that has reached the growth phase,
Add a methanol solution of α ₂ -rhodomycinone or β-isoromycinone to a final purulent density of 10 to 200μ.
g/ml and continue culturing for an additional 15 to 72 hours to complete microbial conversion. In order to suppress foaming during fermentation, antifoaming agents such as Adekanol (trademark of Asahi Denka Kogyo Co., Ltd.) and silicone (trademark of Shin-Etsu Chemical Co., Ltd.) can be added as appropriate. To collect the compound of the present invention, the culture solution is separated into bacterial cells and liquid, and the crude pigment containing the compound is extracted and purified from the bacterial cells and liquid. Acetone, methanol, chloroform, ethyl acetate, toluene, dilute mineral acid, acidic buffer, etc. are used for extraction. For purification, columns and thin layer chromatography using silica gel, cross-linked dextran gel (e.g. Cephadex LH-20; Pharmacia trademark), weakly acidic ion exchange resin, etc., and liquid chromatography using an appropriate molten soot are used. This can be advantageously carried out by combining conventional methods such as , countercurrent distribution method, etc. For example, the product is separated from unconverted aglycones by gel filtration of the crude extract on a cross-linked dextran gel (Sephadex LH-20) column, followed by a thin layer of preparative silica gel (PF ₂₅₄ , Merck & Co.). The purified compound of the present invention can be easily obtained by repeating the chromatography while changing the soot solution. Since the compound of the present invention has a basic group, a sugar residue L-rhodosaminyl group having a dimethylamino group, it can be obtained as a free base or as an acid addition salt with an inorganic or organic acid. The free base can be prepared using a non-toxic acid such as sulfuric acid, hydrochloric acid, or
Recovered as addition salts with bromic acid, nitric acid, phosphoric acid, acetic acid, propionic acid, maleic acid, citric acid, succinic acid, tartaric acid, fumaric acid, glutamic acid, pantothenic acid, lauryl sulfonic acid, methanesulfonic acid, naphthalene sulfonic acid, etc. be done. That is, the formation of an addition salt can be obtained by reacting the free base with the above-mentioned non-toxic acid in a suitable solvent and lyophilizing it, or by precipitation using a solvent in which the acid addition salt is only slightly soluble. I can do it. The usefulness of the compound of the present invention obtained as described above was tested by the following test method. 1 Inhibition of growth of cultured mouse leukemia cells (L1210) and inhibition of acid synthesis 5 × 10 ⁴ L1210 cells were added to RPMI1640 medium (Rothwell Park Institute 1640) containing 20% calf serum.
at the same time, the compound of the present invention was inoculated at 0.1 and ml.
It was added at a concentration of 0.5 μg/ml and cultured at 37°C in a carbon dioxide gas incubator for 24 hours to determine the 50% growth inhibition concentration relative to the control group. Furthermore, 10% of the above L1210 cultured cells
5×10 ⁵ cells/ml to RPMI1640 medium containing calf serum
After culturing in a carbon dioxide incubator at 37°C for 1 to 2 hours, the compounds of the present invention were added at various concentrations, and 15 minutes later, ¹⁴ C-uridine (0.05 μCi /ml) and ¹⁴ C-thymidine (0.05 μCi/ml) were added, and the cells were incubated at 37°C for 60 minutes. Add 10% trichloroacetic acid solution to the reaction solution,
At the same time as stopping the reaction, acid insoluble matter is precipitated,
After washing three more times with 10-5% trichloroacetic acid, it was dissolved in formic acid, the radioactivity in the acid-insoluble material was measured, and the 50% uptake inhibition concentration was determined from the radioactivity uptake rate relative to the control group. 2 Acute toxicity to mice The compound of the present invention was added to 0.1M acetate buffer (PH4.5).
The LD ₅₀ value was determined by dissolving the solution in 0.25 ml and administering the solution intraperitoneally to CDF mice. The above results are shown in Table 1 below.

[Reference example]

Preparation of aglycones used as substrates The substrate aglycones used in the present invention can be prepared as follows. Actinomyces rosevoiolacens, a known bacterium that produces roseorubicin A and B
IFO13081 bacteria were grown in a seed medium (100ml/500ml) consisting of 0.3% yeast extract, 1% soluble starch, and pH 7.0.
Shaking culture was carried out for 3 days in an Erlenmeyer flask), and then 4%
Sucrose, 2.5% soybean flour, 0.1% yeast extract, 0.25% salt, 0.32% calcium carbonate, 0.0005% _CuSO4 .
7H ₂ O, 0.0005% MnC _{2.4H 2} O and 0.0005%
Dispense 50 ml of fermentation medium consisting of ZnSO ₄ 7H ₂ O, pH 7.4, inoculate 1 ml each into 250 sterilized 500 ml Erlenmeyer flasks, and place on a rotary shaker (210 rpm).
Culture with shaking at 28°C for 5 days. The obtained fermentation broth is separated into bacterial cells and supernatant by centrifugation.
Extract with acetone, concentrate, and re-extract with chloroform. The supernatant is extracted with chloroform in Step 2, mixed with the extract from the bacterial cells, and concentrated to dryness to obtain a crude substance. Dissolve this in 70 ml of methanol, remove insoluble matter by centrifugation, apply the supernatant to a Sephadex LH-20 column (see above, φ4.0 x 40 cm), elute with methanol, and collect the glycoside fraction that flows out first. Concentrate to dryness. Add 200ml of 0.3N hydrochloric acid to this and heat at 85℃ for 1 hour to hydrolyze. Total amount
Extract with 500 ml of chloroform and concentrate to dryness to obtain a crude aglycone substance containing γ-rhodomycinone, β-rhodomycinone, β-isorodomycinone and α ₂ -rhodomycinone. This was developed on a thin layer for preparation (60PF ₂₅₄ , manufactured by Merck & Co.) using a solvent system of chloroform:methanol (20:1).
β-Isodomycinone with an Rf value of 0.24, and
The α ₂ -rhodomycinone moiety showing an Rf value of 0.21 was scraped off, and chloroform:methanol (5:
1) Elute with the mixed solution, concentrate to dryness, and elute with methanol to a Sephadex LH-20 column (φ25 cm x 50
Final purification was performed using cm) to obtain 30 mg of β-isoromycinone and 68 mg of α ₂ -rhodomycinone. Next, the present invention will be explained in more detail with reference to Examples. Example 1 Soluble starch 1.5%, glucose 1%, soy flour 1
%, yeast extract 0.1%, salt 0.3%, dipotassium phosphate 0.1%, magnesium sulfate (MgSO ₄ 7H ₂ O)
0.1%, copper sulfate (CuSO ₄・5H ₂ O) 0.0007%, iron sulfate (FeSO ₄・7H ₂ O) 0.0001%, manganese chloride (MnC ₂・4H ₂ O) 0.0008%, zinc sulfate (ZnSO ₄・7H ₂ O) medium consisting of 0.0002%, PH7.4,
Dispense 100 ml of Streptomyces galilaeus into a sterilized 500 ml Erlenmeyer flask.
galilaeus) strain KE303 was inoculated from agar culture on a slant, and cultured with shaking on a rotary shaker at 28°C for 48 hours to prepare seeds. Next, inoculate 1 ml of the above seed culture into 1000 sterilized 500 ml Erlenmeyer flasks by dispensing 50 ml of fermentation production medium with the above culture medium composition increased to 2% soybean flour and 0.2% yeast extract. Shaking culture was performed at 28°C on a rotary shaker (210 rpm) for 17 hours. In addition to this, β-isolodomycinone or α ₂ −
0.5 ml of a methanol solution of rhodomycinone (2 mg/ml) was added per flask (final concentration: 20 μg/ml; total amount of each: 360 mg), and the culture was continued for an additional 24 hours. In order to check the conversion production rate of each converted product β-isolodomycin or α ₂ -rhodomycin at the end of fermentation, 5 ml of the culture solution was collected, and 5 ml of a chloroform-methanol (3:2) mixture was added thereto. The mixture was shaken and stirred using a thermomixer, and the product was extracted into the chloroform layer. After concentrating this to dryness,
Dissolve it again in 0.2 ml of chloroform, spot 20μ of it on a thin layer of silica gel F ₂₅₄ (manufactured by Merck & Co., Ltd.), develop it with a developing solvent of chloroform:methanol (10:1) and air dry. The spots of rhodomycinone RDC and α ₂ -rhodomycinone RDC exhibiting an Rf value of 0.30 were quantified using a Shimadzu thin layer chromatography scanner (Model CS-910) at a wavelength of 490 nm. As a result, 80% of both added anthracyclinones
The above has been converted, and β-isorodomycinone
The total amounts of RDC and α ₂ -rhodomycinone RDC produced were 230 and 185 mg, respectively. The main culture solution (18) was collected, the bacterial cells were obtained by centrifugation, and the product was extracted with acetone (2). After concentrating the acetone extract to 1/3 under reduced pressure, the product was dissolved in approximately 1.5 ml of chloroform and concentrated to dryness to obtain a crude extract. Example 2 Each of these crude converted extracts was dissolved in 50 ml of methanol, and after removing insoluble matter by centrifugation, the supernatant was separated into separate Sephadex LH-20
It was added to a column (40 x 5.0 cm) and eluted with methanol. Separate each of the first red-purple pigment categories,
After concentrating to dryness, each was dissolved in a small amount of chloroform and spotted in a linear manner 1.5 cm above the lower end of a preparative silica gel thin layer (60PF ₂₅₄ , manufactured by Merck & Co., Ltd.) (50 sheets), and chloroform: methanol (100: 10). The dye band consisting of β-isorodomycinone RDC or α ₂ -rhodomycinone RDC was scraped off, extracted with approximately 150 ml of a chloroform:methanol (5:1) mixture, and concentrated to dryness. Next, each concentrate was dissolved in 20 ml of methanol,
Sephadex LH-20 column (40 x 4
cm), and the relevant fractions were pooled and concentrated to dryness. Add 30% of each contents to 0.1M acetate buffer (PH3.5)
ml, and after removing a slight amount of insoluble matter by centrifugation, 10 ml of toluene was added to the supernatant and the mixture was shaken and stirred. After standing still, the toluene layer that separates is removed, and this operation is repeated once more.The aqueous layer is separated and PHed with 4N caustic soda at low temperature (10℃).
The mixture was neutralized to 6 to 7, and then extracted with chloroform. After chloroform extraction, 1/3 amount of 0.01 MEDTA
(PH6.0), then water, dried over Glauber's salt, and concentrated under reduced pressure. Excessive n-hexane was added to the concentrated solution to form a dark red precipitate, which was accumulated by over-operation and then vacuum dried to yield 108mg and 86mg of pure β-isolodomycinone RDC and _α2 -rhodomycinone RDC, respectively. Obtained. The physical and chemical properties of β-isoromycinone RDC and α ₂ -rhodomycinone RDC obtained by the present invention are shown below. β-Isolodomycinone RDC Shape Red-purple powder Melting point 123-127℃ Molecular weight 802 Elemental analysis: C ₄₀ H ₅₁ NO ₁₆ Calculated value % 59.85 6.36 1.74 31.92 Experimental value 59.55 6.18 1.76 −

【table】

[Table] IR (KBr) cm ^-1 : 3400, 2920, 1720, 1660, 1590, 1400,
1370, 1300, 1240

[Table] α ₂ - Rhodomycinone RDC Shape Red powder Melting point 128-135℃ Molecular weight 786 Elemental analysis: C ₄₀ H ₅₁ NO ₁₅ Calculated value % 61.07 6.49 1.78 30.53 Experimental value 60.53 6.62 1.80 − Specific rotation [α] ²³ _D +96゜ (c0.04, MeOH)

[Table] IR (KBr) cm ^-1 : 3400, 2930, 1730, 1600, 1460, 1400, 1280

【table】

Claims (1)

[Claims] 1. Structural formula In the formula, when R ¹ represents a hydroxyl group, R ² represents the formula A novel anthracycline antibiotic and its acid addition salt represented by the following sugar residue, in which R ³ represents a hydrogen atom; and when R ¹ and R ² represent a hydrogen atom, R ³ represents the above-mentioned sugar residue.